Sistemas de valoración de la prueba en la Legislación

2. Aspectos doctrinarios de la estafa

2.6. Sistemas de valoración de la prueba en la Legislación

Pharmacokinetics of intravesical gemcitabine – A preclinical study in pigs ¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯¯

study, because the urogenital tract of the pig closely resembles the human urogenital system4. Nevertheless, male pigs were not suitable because the shape of the penis and the preputial diverticulum make it impossible to catheterize transurethrally. The pigs were divided in 3 groups of 5 F2 pigs (F2 pigs are obtained by crossing a York male pig and a F1 female pig; the F1 pig is a cross-breeding between an English pig and a Large White pig). The pigs were housed in special swine stainless steel battery cages and fed with universal swine food (Hendrix UTD B.V., Boxmeer, The Netherlands). The first two groups received one intravesical instillation with 175 mg and 350 mg gemcitabine (provided by Lilly, Houten, the Netherlands) in 50 cc 0.9% (10 French Foley catheter). In the same instillation 10 mg 5- Bromo-2’-deoxyuridine (BrdUrd, Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands) was dissolved. The catheter was clamped and the instillation was maintained into the bladder for 2 hours, after which the bladder was emptied. Blood samples were taken for pharmacokinetics by puncturing the jugular groove, near the manubrium. In this way blood was collected from the cephalic vein, the internal or external or communal jugular vein depending on the puncture site, angle and depth. In these first two groups blood was taken before instillation of gemcitabine, 30, 60 and 120 minutes after the start of instillation, and again 15, 30, 60 and 120 minutes after emptying of the bladder. Blood samples for gemcitabine measurement were drawn in tubes, to which 10 µl of THU (1 mg tetrahydrouridine/ml phosphate buffered saline; Calbiochem, San Diego, CA, USA) were added to prevent ex vivo degradation of gemcitabine by cytidine deaminase in the serum. The whole procedure was done under general anaesthesia. For premedication one shot azaperon (4 mg/kg) i.m. was used. Sedation was induced with a mixture of midazolam 0.45 mg/kg i.v. and atropine 0.045 mg/kg i.v. and maintained with a mixture of ketamine 10 mg/kg/h i.v. and midazolam 0.35 mg/kg/h i.v. The well being of the animals was observed by experienced staff after the procedure and 24 hours after the instillation the animals were euthanized and cystectomized and the bladder was investigated histologically. The third group received 350 mg gemcitabine intravesically in 50 cc 0.9% saline weekly for 6 weeks. The conditions were the same as in the first two groups. The procedure was also done under general anaesthesia. The instillation was left in place for two hours, but blood was only withdrawn 60 minutes after instillation, again for measurement of gemcitabine, white blood cell and thrombocyt count. Animals were observed for clinical signs of toxicity. The animals were euthanized 24 hours after the last instillation; histological examination of the bladder wall was performed.

Chapter 4

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Pharmacokinetics

In order to evaluate the systemic absorption of gemcitabine before, during and after the administration of intravesical gemcitabine, blood samples were collected in heparinised tubes and in CPT-tubes (Cell Preparation Tube with Sodium Citrate, Becton Dickinson Vacutainer Systems, Alphen aan den Rijn, The Netherlands).

After blood sample collection the heparine-tubes (each containing 10 µl of THU (1 mg tetrahydrouridine/ml phosphate buffered saline) were inverted to mix the blood, with the anticoagulant and the THU. Thereafter, the tubes were immediately placed on ice and transferred to the lab for preparation. Centrifugation of the tubes took place for 5 minutes at 4000 rpm at 4°C. The top layer (plasma) was transferred with a pipette into another tube. These tubes were frozen, stored at -20°C, and further processed in Amsterdam. The CPT- tubes were inverted after blood collection to mix the anticoagulant additive with blood (these tubes do not contain THU). The tubes were stored at room temperature until centrifugation. The blood samples were remixed immediately prior to centrifugation by gently inverting the tube and then centrifuged for 30 minutes at 3200 rpm at normal room temperature. After centrifugation the whitish layer just under the plasma layer was collected with a pipette together with the plasma layer to be transferred to a new tube. The procedure was repeated and again the two above-mentioned layers were transferred to a new tube. To this tube PBS (4°C) was added to a volume of 10 ml. 20 µl were used to count the cells with a counting chamber. The rest was centrifuged for 25 minutes at 2500 rpm at 4°C. As much supernatant as possible was aspirated without disturbing the cell pellet and the cell pellet was transferred to a 1,5 ml Safelock tube. Again centrifugation took place for 5 minutes at 13000 rpm at 4°C. The supernatant was removed and the cell pellet immediately frozen in liquid nitrogen and stored at -80°C until analysis of nucleotides and 2’2’-difluoro-2’deoxycytidine-triphosphate (dFdCTP), the active metabolite of gemcitabine.

All samples were sent as one batch to the VU University Medical Centre in Amsterdam, The Netherlands. Plasma levels of gemcitabine and its metabolite 2’2’-difluorodeoxyuridine (dFdU) were measured with a validated ion-pair reversed-phase HPLC assay using an Aqua column (150 x 4.6 mm, 3 µm) (Phenomenex, Bester, Amstelveen, The Netherlands) as described previously5. The concentrations of normal nucleotides and of dFdCTP in white

described previously5. Blood samples for leukocytes and thrombocyte measurement were collected in K3E 15% tubes and sent to our clinical chemical laboratories for determination of levels.

Results

One instillation of 175 and 350 mg gemcitabine was well tolerated, as well as the course of 6 weekly instillations with 350 mg gemcitabine. The animals showed no signs of deterioration of their well-being. Pharmacokinetic studies showed that both gemcitabine and its metabolite dFdU were not measurable in plasma, both in samples collected at the single administration and in samples collected after repeated administration. Since the active metabolite of gemcitabine, dFdCTP might accumulate in white blood cells even after discontinuation of the instillation, we also evaluated whether this metabolite would be measurable in white blood cells. However, in none of the white blood cells dFdCTP was measurable. Since we observed previously5,6 that gemcitabine can have profound effects on normal nucleotide pools we also evaluated whether the concentrations of normal nucleotides and their ratios was affected by gemcitabine instillation. E.g. the ratio CTP/UTP was related to the efficacy of gemcitabine in a cell line6. However, both the ATP/ADP ratio and that of CTP/UTP did not

change during instillation and their ratios were comparable to that published previously7. These data demonstrate that in healthy pigs systemic absorption of gemcitabine would be negligible at least during the 4 hours after beginning of instillation.

In group 3 peripheral blood counts displayed no signs of immunosuppression (figures 1 and 2). Both leukocyte counts and erythrocyte counts remained within the normal range.

The resected bladder showed no macroscopic abnormalities such as oedema or erosions. Histology of the bladder wall showed in all cases normal bladder histology, without mucosal atypia and detrusor changes. Only in some cases mild signs of inflammatory response (subepithelial leukocyte infiltration) and epithelial cellular and nuclear enlargement were seen, mainly in group 3 after 6 instillations (figures 3-5).

Chapter 4

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Figure 3: Histological appearance of a normal pig bladder.

Figure 4: Pigs bladder after one instillation with 350 mg gemcitabine in 50cc 0.9% NaCl. Slight thickening of the urothelial layer can be seen.

Figure 5: Pigs bladder after 6 instillations with 350 mg gemcitabine in 50cc 0.9% NaCl. Subepithelial leukocyte infiltration as can be seen in case of infection (left). Thickening of the urothelial layer can be seen, with some cellular and nuclear enlargement (right).

Chapter 4

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Discussion

As pointed out in the introduction, there are several arguments to look for new drugs for intravesical use in case of superficial bladder cancer. Currently used chemotherapeutic drugs are very safe, but lack significant prevention of recurrences on the long run, and have no impact on progression to muscle invasive disease. Intravesical BCG, on the other hand, is more effective in reducing the recurrence rate, even seems to reduce progression, but is significantly more toxic, with even deaths having been described.

Gemcitabine (2’,2’-difluorodeoxycytidine, dFdC) is transported into the cell and phosphorylated (dFdC-triphosphate, dFdCTP) prior to incorporation into DNA and RNA, which causes inhibition of growth activity and mediates apoptosis8. Gemcitabine is deactivated by deamination into 2’,2’-difluorodeoxyuridine (dFdU) and transported out of the cell. Gemcitabine was first screened for antiviral activity in the eighties, but appeared to have remarkable activity in solid tumours, such as ovarian and non small-cell lung cancer. The first preliminary report on the use of gemcitabine in advanced bladder cancer was published in 19949. In subsequent single agent trials promising response rates and acceptable toxicity were reported, even as second line10-12_{. After these single-agent studies several phase II reports} were published combining gemcitabine with other drugs, again with mild toxicity, good tolerance and promising response rates between 54 and 68%13-17. Finally, a large phase III study in 405 patients with locally advanced or metastatic TCC compared gemcitabine and cisplatin (GC) versus methotrexate, vinblastine, doxorubicin and cisplatin (MVAC)18. The results suggest a better safety profile and tolerability of GC, and similar survival advantage, although the trial was not designed as an equivalence trial.

Gemcitabine has several features that make it an attractive alternative chemotherapeutic agent for intravesical use in patients with TCC. As has been explained above, the single-agent activity against TCC is significant. The molecular weight of gemcitabine (299,66) is high enough to assume there will be no systemic absorption. Furthermore, the systemic toxicity profile is mild, so in case of systemic absorption the consequences would be minimal.

Two phase I studies with intravesical gemcitabine have been published to date19,20. Both studies conclude that gemcitabine has substantial activity, even in BCG refractory patients. Local side effects are minimal, systemic side effects minimal in case of one instillation weekly, tolerable in case of a twice-weekly instillation schedule. One other animal study has

beagle dogs. Three groups of 2 dogs received 100 mg, 350 mg (equivalent to the 1000 mg/m2 human dose) and 1000 mg gemcitabine in 50 ml normal saline intravesically, three times one hour weekly for 4 weeks. Two dogs also received 350 mg intravenously over a 1 hour period. Animals were observed clinically, and blood samples for pharmacokinetics and peripheral blood counts were taken three times weekly. The dogs were euthanized, and a full necropsy was performed at days 1 and 14 after the last dose. Both 100 mg and 350 mg intravesically were tolerated without clinical signs of side effects. With the 350 mg dose a slight drop in platelet and white cell count was seen towards the end of the 4-week treatment period (12 instillations) with quick recovery after cessation of therapy. At necropsy no toxic effects were observed, in particular not in the bladder or bone marrow. Initially two dogs were treated with 350 and 3500 mg intravesically, and two additional study dogs were treated with 1000 mg. Both the 1000 mg as well as the 3500 mg dose caused severe problems: clinical toxicity, myelosuppression, high serum levels of gemcitabine and multi-organ toxicity on necropsy. In all doses gemcitabine could be measured in serum, with lower serum levels corresponding with lower intravesical concentrations. The serum level after the intravesical use of 350 mg (approximately 30 µM) was significantly lower than the serum level after intravenous administration of a similar dose (approximately 80 µM), similar to concentrations observed at high doses gemcitabine in humans5_{. However, serum levels after intravesical use were} measurable up to 8 hours after administration, corresponding with a significant longer half life time of 328 minutes as compared to 99 minutes after intravenous use, both in the 350 mg dose (p<0.001). The authors concluded that, although all doses of gemcitabine were absorbed systemically, it was possible to deliver a clinically active dose without bladder or systemic toxicity. Our results in pigs support part of the results found in dogs. We also did not find any problems with the animals well being, bladder toxicity or systemic toxicity. A major difference, however, is the fact that in our pig model no systemic absorption of gemcitabine could be measured. This can be explained by the differences in instillation schedules. Our instillation schedule (once weekly) was much less frequent than in the dog study (3 times weekly), although instillation time was 2 hours as compared to 1 hour in the dog study. It is well known that intravesical chemotherapy causes some chemical cystitis with hyperaemia. Twelve instillations within three weeks might therefore, very well result in drug absorption, especially since this was seen mostly towards the end of the instillation schedule. The difference in animal size (pigs are bigger) with the same absolute dose in both studies does not seem to be relevant.

Chapter 4

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From a theoretical point of view, gemcitabine is worth investigating for the use in superficial bladder cancer. In our animal experiment it was safe in all aspects studied, without systemic absorption. A subsequent phase I human study, studying 1000, 1500 and 2000 mg gemcitabine in 50 ml saline for intravesical use, will be discussed in chapter 6 of this thesis.

In document La Prueba Testimonial en el Delito de Estafa (página 69-74)